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TCR signal strength controls thymic differentiation of discrete proinflammatory γδ T cell subsets

Nature Immunology volume 17, pages 721–727 (2016)Cite this article

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Abstract

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g+/− Cd3d+/− (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes of αβ thymocyte subsets, but impaired differentiation of fetal Vγ6+ (but not Vγ4+) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122+ NK1.1+ γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ+ γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

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Figure 1: γδ T cells from CD3DH mice show reduced TCRγδ expression and signaling.
Figure 2: CD3DH mice show impaired differentiation of IL-17+ and IFN-γ+ γδ T cells within selective windows of TCR Vγ usage.
Figure 3: Transcriptional signatures of TCR signal strength in γδ thymocytes.
Figure 4: CD3DH mice lack IFN-γhiCD122+NK1.1+ thymocytes, which are rescued by CD3 crosslinking in vivo.
Figure 5: CD3DH mice show reduced peripheral IFN-γ+ γδ T cells and are resistant to cerebral malaria.

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Acknowledgements

We thank K. Serre, J. Martins, N. Schmolka, A. Amorim, V.Z. Luís and M.M. Mota (all at iMM Lisboa); and B. Garcillán, D. de Juan, M. Mazariegos, M. Sanz-Rodríguez and S. Diaz-Castroverde (Complutense University) for help and advice; A. Hayday (King's College London) and P. Pereira (Pasteur Institute) for insightful discussions; and the staff of the animal and flow cytometry facilities at iMM Lisboa and Complutense University for technical assistance. This work was funded by the European Research Council (StG_260352 and CoG_646701 to B.S.-S.); MINECO (SAF2011-24235 and BES-2012-055054 to J.R.R.), CAM (S2010/ BMD-2316/2326 to J.R.R.) and the Lair Foundation (2012/0070 to J.R.R.); and FIS PI11/02198 and MINECO SAF2014-54708-R to E.F.-M.

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  1. Edgar Fernández-Malavé and Bruno Silva-Santos: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology, School of Medicine, Complutense University, Madrid, Spain

    Miguel Muñoz-Ruiz, José R Regueiro & Edgar Fernández-Malavé

  2. Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal

    Miguel Muñoz-Ruiz, Julie C Ribot, Ana R Grosso, Natacha Gonçalves-Sousa, Ana Pamplona & Bruno Silva-Santos

  3. Blizard Institute, Barts and The London School of Medicine, Queen Mary University of London, London, UK

    Daniel J Pennington

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Contributions

M.M.-R., B.S.-S., E.F.-M., J.R.R. and D.J.P. designed research; M.M.-R., J.C.R., A.R.G., N.G.-S. and A.P. performed experiments; B.S.-S., E.F.-M. and J.R.R. supervised research and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 γδ and αβ T cell development in CD3 mutant mice.

(a) Flow cytometry showing the CD3ɛ vs TCRδ phenotype of thymocytes from adult WT or CD3SH (single heterozygote for CD3δ or CD3γ) mice (n = 3 per group). Numbers within gated areas indicate % cells in each throughout. (b) Left, representative CD3ɛ vs TCRδ phenotype of thymocytes from the indicated adult mice. Numbers as in (a). Right, CD3ε and TCRδ expression (MFI) in gated TCRδ+ thymocytes and CD3+ thymocytes, respectively, from the indicated adult mice (n = 3 per group). Numbers indicate MFI (c) Gating strategy for CD3+ TCRδ+ γδ T cell identification. Doublets and dead cells were excluded. (d, e) Comparative analysis of TCRβ+CD1d+ NKT cells (d) and CD4+Foxp3+ Treg cells (e) in WT and CD3DH mice. Numbers in c, d, and e indicate percentage of cells within the marked region.

Supplementary Figure 2 γδ T cells from CD3DH mice show impaired TCRγδ signaling.

(a) Flow cytometry of phosphorylated AKT (empty histograms) in sorted TCRδ+CD3+CD27+ lymph node cells from the indicated mice, after 5 min stimulation with PMA plus ionomycin; filled histograms represent staining with isotype-matched control antibody. (b) Intracellular calcium mobilization in TCRδ+CD3+CD27+ lymph node cells obtained from the indicated mice, after 5 min stimulation with 10 µg/ml biotinylated anti-CD3ɛ mAb followed by TCR crosslinking with 10 µg/ml streptavidin, and then assessed over 5 min, followed by 2 min stimulation with PMA plus ionomycin.

Supplementary Figure 3 Contributions of WT or CD3DH progenitors to γδ and CD4+ thymocytes in mixed bone marrow chimeras.

Thy-1.1+ (WT-derived) vs Thy-1.2+ (CD3DH-derived) fractions within TCRδ+CD3+ thymocytes (a) and CD4+CD3+ thymocytes (b) from 1:9 mixed WT:CD3DH BM chimeras. Each symbol indicates one individual host, either RAG2−/− (squares) or TCRδ−/− (triangles). *P < 0.01, **P < 0.001 (Student’s t-test). Data are from three independent experiments.

Supplementary Figure 4 Transcriptional signatures of Vγ-based γδ thymocyte subsets.

Relative expression of Egr3, Id2, Id3 and Gp49b (a) or Sox4, Sox13, Bcl11b and Blk (b), in arbitrary units normalized to the housekeeping gene hprt, in sorted Vγ1+, Vγ4+ or Vγ1−Vγ4− thymocytes from either WT or CD3DH E18 embryos. Data shown are the mean +/− s.d.

Supplementary Figure 5 CD4+ T cells from CD3DH mice differentiate normally into TH1 cells.

(a) Flow cytometry of intracellular IFN-γ expression of WT (top) and CD3DH (bottom) TCRβ+CD4+ splenocytes incubated for 4 days without stimuli (left panels) or on plate-bound α-CD3ɛ and α-CD28 mAb (5ng/ml each), without (middle panels) or with (right panels) Th1-polarizing cytokines IL-2 (10 ng/ml) and IL-12 (50 ng/ml) plus anti-IL-4 (10 µg/mL) neutralizing antibody. Numbers indicate % of cells in each quadrant. Data are representative of two independent experiments.

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Muñoz-Ruiz, M., Ribot, J., Grosso, A. et al. TCR signal strength controls thymic differentiation of discrete proinflammatory γδ T cell subsets. Nat Immunol 17, 721–727 (2016). https://doi.org/10.1038/ni.3424

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